EP1004668A2 - Protéine régulatrice pKe 83 de keratinocytes humains - Google Patents

Protéine régulatrice pKe 83 de keratinocytes humains Download PDF

Info

Publication number
EP1004668A2
EP1004668A2 EP99122909A EP99122909A EP1004668A2 EP 1004668 A2 EP1004668 A2 EP 1004668A2 EP 99122909 A EP99122909 A EP 99122909A EP 99122909 A EP99122909 A EP 99122909A EP 1004668 A2 EP1004668 A2 EP 1004668A2
Authority
EP
European Patent Office
Prior art keywords
seq
nucleic acid
pke
protein
sequence
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Granted
Application number
EP99122909A
Other languages
German (de)
English (en)
Other versions
EP1004668A3 (fr
EP1004668B1 (fr
Inventor
M. D. Pd Dr. Med. Kramer
Michael Dr. Bechtel
Jeanette Dr. Rer. Nat. Reinartz
Birgit Dr. med. Schäfer
Reinhard Pd Dr. Rer.Nat. Wallich
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Kramer Michael
Original Assignee
Kramer Michael
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from DE19856301A external-priority patent/DE19856301C1/de
Application filed by Kramer Michael filed Critical Kramer Michael
Publication of EP1004668A2 publication Critical patent/EP1004668A2/fr
Publication of EP1004668A3 publication Critical patent/EP1004668A3/fr
Application granted granted Critical
Publication of EP1004668B1 publication Critical patent/EP1004668B1/fr
Anticipated expiration legal-status Critical
Expired - Lifetime legal-status Critical Current

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4702—Regulators; Modulating activity

Definitions

  • the invention relates to an isolated polypeptide which is naturally present in Keratinocytes occurring and in the activated state of the keratinocytes expressed protein is similar or similar (i.e. in function and Effect is the same). It also relates to an isolated nucleic acid, the one encodes such a typical polypeptide or protein for human keratinocytes, and the use of this polypeptide and nucleic acid for demonstrative, especially diagnostic, and / or for therapeutic Purposes, and reagents using at least one of these Molecules are produced, in particular recombinant vector molecules and Antibody.
  • Targets a point of attack for a (specific) Influencing cellular metabolism - especially under medical or cosmetic aspects - could serve is narrowly limited in epidermal keratinocytes.
  • the object of the present invention is therefore to create new target structures to provide epidermal keratinocytes that act as a target for Diagnostics, therapeutics, cosmetics or in general for influencing can serve the cellular metabolism.
  • polypeptide or protein of the type mentioned at the outset which is upregulated in the case of activated keratinocytes, ie is expressed or produced more and is kept at a higher concentration level, and which is contained in the sequence listing SEQ ID NO: 3 or SEQ ID NO: 4 or SEQ ID NO: 6 or SEQ ID NO: 8 amino acid sequence shown or an allele or derivative of this amino acid sequence resulting therefrom by amino acid substitution, deletion, insertion or inversion.
  • the polypeptide with the amino acid sequence according to SEQ ID NO: 3 or SEQ ID NO: 4 or SEQ ID NO: 6 or SEQ ID NO: 8 is also referred to below as protein "pKe # 83".
  • a further solution to the stated problem consists in providing an isolated nucleic acid which encodes a protein which is similar or similar to a protein which occurs naturally in human keratinocytes and which is expressed in the activated state of the keratinocytes, and which is either similar to that in the sequence listing SEQ ID NO: 1 or the nucleotide sequence shown in the sequence listing SEQ ID NO: 7 or a complementary nucleotide sequence or a partial sequence of one of these two nucleotide sequences or a nucleotide sequence hybridizing wholly or partially with one of these two nucleotide sequences, with SEQ ID NO: 1 and SEQ ID NO: 7 can also be "U" instead of "T".
  • This group of nucleic acids or nucleotide sequences according to the invention also includes, in particular, splice variants (for example SEQ ID NO: 2 or SEQ ID NO: 5 ) and sense or antisense oligonucleotides which correspond to those in the sequence listing SEQ ID NO: 1 or with the hybridize nucleotide sequence shown in the sequence listing SEQ ID NO: 7 , are preferably identical to or (partially) complementary to it.
  • Two preferred splice variants of the nucleotide sequence according to the invention according to SEQ ID NO: 1 and SEQ ID NO: 7 are shown in the sequence listing SEQ ID NO: 2 and SEQ ID NO: 5 .
  • the invention consequently also includes proteins or polypeptides of the type mentioned at the outset, which have an amino acid sequence which results from such a splice variant, in particular from the splice variant of an mRNA which corresponds to the sequence protocol SEQ ID NO: 1 or to the nucleotide sequence specified in the sequence listing SEQ ID NO: 7 is identical or completely or partially complementary thereto.
  • the sense and antisense oligonucleotides according to the invention include in each case at least 6, preferably 8-25 nucleotides.
  • hybridized refers to the hybridization methods known in the prior art under customary, in particular under highly stringent hybridization conditions.
  • the person skilled in the art chooses the specific hybridization parameters on the basis of the nucleotide sequence used and his general specialist knowledge (see: Current Protocols in Molecular Biology, Vol. 1, 1997, John Wiley & Sons Inc., Suppl. 37, Chapter 4.9.14).
  • the present invention also includes such nucleotide sequences that hybridize under stringent conditions.
  • hybridize or “hybridization” according to the present invention is used as in Sambrook et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor, Laboratory, Press, 1989, 1,101 to 1,104 .
  • the nucleic acid (s) according to the invention can either be from a natural source as well as synthetic or semi-synthetic become. In practice, execution as a cDNA has proven particularly useful.
  • polypeptide which has the amino acid sequence according to SEQ ID NO: 3 or SEQ ID NO: 8 and which is encoded by the nucleic acid shown in the sequence listing SEQ ID NO: 1 or SEQ ID NO: 7 , and which is hereinafter referred to as the protein "pKe # 83 "is upregulated in human epidermal keratinocytes, namely expressed (produced) to an increased extent and kept at a concentration level which is significantly higher than in the initial state when these cells are in the" activated "state, ie in the state of proliferation and / or migration, eg after an accident-induced skin injury or in the autoimmunologically triggered bullous dermatoses "Pemphigus vulgaris” (triggered by autoantibodies against desmosomes) and “Bullous pemphigoid” (triggered by autoantibodies against hemidesmosomes).
  • the activated state of the human epidermal keratinocytes is also expressed in an increased expression of the known activation markers uPA (urokinase-type plasminogen activator) and uPA-R (receptor for urokinase-type plasminogen activator) compared to the resting state (initial state) and can be qualitatively and based on these markers quantitatively detected (see: Shufer, et al., 1996: Dispase-mediated basal detachment of cultured keratinocytes induces urokinase-type plasminogen activator (UPA) and its receptor (uPA-R, CD87), Exp. Cell Res. 228 , pp. 246-253).
  • UPA urokinase-type plasminogen activator
  • uPA-R receptor for urokinase-type plasminogen activator
  • the protein pKe # 83 has a so-called prenyl group binding site ("CAAX Box "). This is a binding site that is associated with a Large number of eukaryotic proteins a post-translational change allowed by attaching a farnesyl or gerany-geranyl group to one Cysteine residue is attached, which removes three amino acids from the C-terminus and where the two amino acids located at the C-terminus in the Are generally aliphatic. Ras proteins and a variety of G proteins have such a CAAX box.
  • the protein "pKe # 83" has a number of putative Phosphorylation sites.
  • the motifs mentioned indicate that the Protein pKe # 83 is involved in signal transduction processes.
  • the invention further relates to recombinant DNA vector molecules which comprise a nucleic acid according to the invention, and which have the ability to Expression of an occurring in human keratinocytes and in activated state of the keratinocytes increased expressed protein, especially the protein pKe # 83, in a prokaryotic or exhibit eukaryotic cell.
  • DNA vector molecules are concerned it is preferably a derivative of the plasmid pUEX-1 and / or the Plasmid pGEX-2T and / or the plasmid pcDNA3.1, since these Vectors have proven to be very suitable in practice.
  • the vector construct pGEX-2T / pKe # 83 according to the one in FIG.
  • a eukaryotic Cell in particular cells come from cell cultures, e.g. Cos cells, in Consider, the cell in question can also be part of a living organism, e.g. a transgenic mouse.
  • the invention therefore also includes transformed host cells, the one contain nucleic acid according to the invention with an activatable Promoter is coupled to these cells naturally or as a result recombination is included, and (as a result) the ability to Expression of an occurring in human keratinocytes and in activated state of the keratinocytes increased expressed protein, in particular the protein "pKe # 83".
  • the invention further relates to the use of an inventive Nucleic acid or a vector molecule according to the invention Production of transgenic mammals, especially mice or rats.
  • the transfectants according to the invention enable research and Development work for the purpose of further elucidation of the the protein "pKe # 83" induced changes in cell morphology and basic cellular functions such as proliferation, adhesion, migration and Differentiation, especially with regard to answering the question, whether the protein "pKe # 83" itself has a "pathogenic” activity.
  • the present invention also relates to a reagent for indirect detection of an occurring in human keratinocytes and in activated state of the keratinocytes increased expressed protein, especially the protein "pKe # 83", whereby this reagent is characterized in that it has at least one nucleic acid according to the invention includes.
  • “for indirect detection” means that in fact the mRNA encoding the protein is directly detected -and thus the protein only indirectly (by means of this mRNA).
  • the protein "pKe # 83" and the polypeptides related to it, ie with the polypeptides shown in the sequence listing SEQ ID NO: 3 or with the amino acid sequence shown in the sequence listing SEQ ID NO: 8 , ie the polypeptides produced by substitution, deletion, insertion and / or Inversion can be derived from the amino acid sequence according to SEQ ID NO: 3 or according to SEQ ID NO: 8 or which have an amino acid sequence which results from a splice variant of an mRNA which corresponds to the nucleotide sequence according to the sequence listing SEQ ID NO: 1 or according to the sequence listing SEQ ID NO: 7 or a partial sequence thereof, which is identical or complementary to it, or at least hybridized, offer diverse application possibilities in the field of dermatological research and development.
  • antibodies against these polypeptides or proteins can be produced which, with appropriate modification, can then be used either as diagnostics or as therapeutic agents or also as cosmetics ("cosmeceuticals").
  • the invention consequently also includes the use of such a protein or polypeptide for the production of a (monoclonal or polyclonal) Antibody against this polypeptide, said antibody itself and likewise its use for diagnostic and / or therapeutic Treatment of dermatological diseases, for cosmetic Treatment of the epidermis and for diagnosis and / or cosmetic Treatment of other tissues expressing the protein "pKe # 83" or organs.
  • sense and / or antisense oligonucleotides are also suitable as active ingredients for pharmacotherapy (cf. G. Hartmann et al. 1998: Antisense oligonucleotides, Inc. ⁇ videblatt 95, No. 24, C1115-C1119) - and moreover as active ingredients with a principle of action that is fundamentally new in pharmacotherapy.
  • the present invention therefore also relates to the use Sense or antisense oligonucleotides according to the invention for diagnosis and / or therapeutic treatment, especially dermatological Diseases, or for cosmetic treatment, especially the Epidermis.
  • nucleic acid consists in the fact that with the help of such Molecule in a "screening" process from a very large number available substances can be selected from those specifically to the nucleic acid or polypeptide in question tie. These substances can then be used as the starting material (lead structure) for the Serve and offer development of pharmacologically usable substances thus the prerequisites for the development of alternative pharmaceuticals for diagnosis and therapy, especially those mentioned at the beginning dermatological diseases and / or other diseases in which the protein "pKe # 83" plays an important role.
  • the invention also relates to the use of a polypeptide according to the invention or a polypeptide according to the invention Nucleic acid for the identification of pharmacologically usable substances, that bind to the polypeptide or the nucleic acid and thereby its or influence their function and / or expression, in particular have an inhibiting or activating effect.
  • This cell culture or this cell culture model is characterized in that it allows keratinocytes to be separated from the resting [uPA - / uPA-R - ] by enzymatic destruction of the cell / matrix contacts, for example by a dispase-induced detachment of the keratinocytes from the culture matrix. convert to the activated [uPA + / uPA-R + ] state.
  • NHEK normal human epidermal keratinocytes
  • NHEK obtained by skin biopsy were trypsinized overnight at 4 ° C. and then using the "Feeder Layer” technique by JG Rheinwald and H. Green (1975, Serial cultivation of strains of human epidermal keratinocytes: the formation of keratinizing colonies from single cells, Cell 6, pp.
  • DMEM Dulbecco's modified Eagle's Medium
  • FCS fetal calf serum
  • adenine hemisulfate insulin, transferrin, triiodothyronine, hydrocortisone, forskolin, epidermal growth factor (EGF) and antibiotics (penicillin, streptomycin and gentamycin) under differentiation conditions, especially increased calcium levels, 37 ° C, cultivated , 7% CO 2 ).
  • EGF epidermal growth factor
  • antibiotics penicillin, streptomycin and gentamycin
  • the incubated cells were converted using the guanidinium-thiocyanate-phenol-chloroform extraction method known in the prior art (see: Chromczynski P. and Sacchi N., 1986: Single-step method of RNA isolation by acid guanidinium-thiocyanate-phenol-chloroform extraction. Anal. Biochem. 162: pp. 156-159 ) the entire RNA was obtained (kit "RNA-Clean" from AGS, Heidelberg). The mRNA was isolated from the total RNA by binding to poly-T coated beads. This mRNA served as the starting material for the next process step of subtraction cloning.
  • mRNA isolated from adherent keratinocyte sheets after the same procedure as described above, except the Deviation that for the duration of the disposition treatment in addition to the Dispase a dispase inhibitor, e.g. Phosphoramidon (100 ⁇ g / ml), applied has been.
  • a dispase inhibitor e.g. Phosphoramidon (100 ⁇ g / ml)
  • a gene bank was created based on the principle of subtraction cloning, which preferably contained the cDNA of the dyshesion-induced genes, i.e. those genes that increase in number after the keratinocyte sheets are detached these (or their cells) were expressed.
  • the mRNA obtained from the cells of the adherent keratinocyte sheets again bound to poly-T coated beads, on these in single-stranded cDNA and then rewritten against the mRNA detached, i.e. non-adherent keratinocyte sheets hybridized.
  • the resulting library was then subjected to a Southern blot procedure with [ 32 P] -labelled cDNA-adherent and non-adherent keratinocyte sheets for the purpose of checking.
  • Those cDNA or rather the host cell clones containing them - here of the E. coli strain MC1061 - which showed a clear upregulation after dyshesion, were then cultivated or propagated overnight at 30 ° C. under normal culture conditions.
  • the plasmid DNA (pUEX1 cDNA) was prepared from these E. coli clones, and the cDNA fragments cut out from the pUEX1 vector were labeled by random priming [ 32 P].
  • the labeled cDNA was used as a probe in Northern blots with RNA from adherent and non-adherent keratinocyte sheets.
  • the clones which contained cDNA which, when used as a probe in the Northern blot method, gave no or only a slight signal with the RNA-adherent keratinocytes, but a clear signal with RNA with non-adherent keratinocyte sheets, were selected for the subsequent sequence of the sequencing process.
  • sequence listing SEQ ID NO: 7 derived an amino acid sequence which are shown in the sequence listing SEQ ID NO: 3 and SEQ ID NO: 8.
  • sequence listing SEQ ID NO: 8 The structural analysis of these amino acid sequences according to sequence listing SEQ ID NO: 3 and SEQ ID NO: 8 with the same Program provided the following information:
  • the protein pKe # 83 has a so-called prenyl group binding site ("CAAX box") and a number of possible phosphorylation sites (9x protein kinase C, 15x casein kinase II, 2x tyrosine kinase according to SEQ ID NO: 3 and 24x protein kinase C, 29x casein kinase II, 5x tyrosine kinase according to SEQ ID NO: 8). These motifs are an indication that the protein pKe # 83 is involved in signal transduction processes.
  • the protein pKe # 83 according to SEQ ID NO: 8 also has several (eight) myristylation sites.
  • rt-PCR polymerase chain reaction after reverse transcription
  • 10 ng of cDNA with 10 ⁇ M primer each were used together with a mixture of heat-stable DNA polymerase, ATP, TTP, GTP, CTP and polymerase buffer (see, for example: Current Protocols in Molecular Biology , Vol. 1, 1997, John Wiley & Sons Inc., Suppl. 37, Chapter 15), here in the example in the form of the commercially available "PCR master mix" from Clontech.
  • the following control tests were carried out: 1.
  • reaction mixture described above with the plasmid pUEX / pKe # 83 instead of the cDNA 1. the reaction mixture described above without the addition of cDNA (“negative control”), 3. the reaction mixture described above Reaction mixture with GAPDH-specific primers (# 302047, Stratagene; "GAPDH control”).
  • a PCR product of the expected size of ⁇ 390 bp was found in the lanes 2, 5 - 8 proven, i.e. pKe # 83-specific mRNA was found in the Keratinocyte sheets (NHEK) at 2, 4 and 8 hours after Dispase-induced detachment and also detected in HaCaT cells.
  • Fig. 1.B shows the result of a GAPDH-specific PCR. The following applies:
  • GAPDH control This GAPDH-specific PCR proves that a negative PCR result in the pKe # 83-specific approach did not affect one Absence of cDNA is due to the fact that in all Reaction batches from T0 - T8 a PCR product of the expected size 600 bp was detectable.
  • Rt-PCR enables the detection of pKe # 83 expression even in cases in which the detection of the pKe # 83 protein is too low Expression level with immunohistological methods, the ELISA or not succeed with immunoblot procedures.
  • Example 3 Production of vector molecules with the ability to Expression of the protein pKe # 83 in prokaryotic or eukaryotic cells, as well as production and cleaning of the recombinant pKe # 83 protein
  • the vector construct pGEX-2T / pKe # 83 was produced according to the vector protocol in FIG. 2 for expression in bacteria ( E. coli DH5 ⁇ ).
  • the vector construct pcDNA3.1 / pKe # 83-FLAG was produced according to the vector protocol in FIG. 3 ) for the purpose of expression in eukaryotic cells (Cos cells).
  • the vector construct pGEX-2T / pKe # 83 was used according to standard protocols for the transformation of E. coli DH5 ⁇ .
  • the pKe # 83-glutathione-S-transferase (GST) fusion protein was expressed in bacteria, the bacterial lysate was examined in immunoblot with anti-GST antibodies, in comparison to lysate from bacteria which were treated with a control plasmid (none GST) were transformed.
  • the pKe # 83 / GST fusion protein was determined by affinity chromatography Purified from bacterial lysates with the help of gluthathione-Sepharose 4B. The Fractions from this purification were then immunoblotted with anti-GST antibodies examined.
  • the pKe # 83 / GST fusion protein had an apparent molecular weight of approx. 90 KDa. This allows the conclusion that the 90 KDa pKe # 83 / GST fusion protein from the GST protein (approx. 26 kDa) and an approx. 60 - 65 KDa large fragment of the protein pKe # 83 exists.
  • the result of this experiment demonstrates the expression of the pKe # 83-FLAG fusion protein in cos cells using the vector construct pcDNA3.1 / pKe # 83-FLAG were transfected.
  • the arrow marks the position of the pKe # 83 protein.
  • Immunohistology With the help of a cryotome, 5 ⁇ m thick frozen sections of tissue from skin biopsies of clinically normal normal skin and Dispase-detached NHEK "sheets" were made at time T0 and T8. These were air dried at room temperature and fixed in 100% acetone (100% methanol, 100% ethanol or 4% paraformaldehyde can also be used instead of acetone). The sections were then treated in accordance with what are known as “blocking methods” in the prior art in order to block non-specific binding sites for the antibody. In the present example, two blocking steps were carried out: (1) blocking with avidin / biotin and (2) blocking with normal serum.
  • the cuts were made in PBS with a Content of 5 ⁇ g / ml rabbit "anti-pKe # 83 IgG" for 1 hour Incubated at room temperature. To remove the unbound antibody the sections were then made in PBS containing 0.2% (Weight / volume) bovine serum albumin washed. The follows Incubation with, for example, a biotin-labeled and against Goat rabbit IgG-directed antibody (diluted 1: 500 in PBS / 0.2% BSA; 30 minutes at room temperature), another Washing step, as well as the application of one with the fluorescent dye Cy3-labeled streptavidins (1: 1000 diluted in PBS / 0.2% BSA).
  • FIG. 8 The results of an immunofluorescence test carried out in this way are shown in FIG. 8 :
  • the rabbit "anti-pKe # 83 IgG” shows weak intracellular and strong cell membrane-associated immunostaining on normal skin sections ( FIG. 8.C ).
  • This result includes the statement that little protein pKe # 83 was present or at least detectable immediately after detachment, but that increased expression had already taken place 8 hours later and consequently significantly higher amounts of protein pKe # 83 could be detected.
  • Enzyme-linked immunosorbent assay To quantify the pKe # 83 protein in complex solutions, a so-called “sandwich” ELISA (FIG. 7) was carried out. For this purpose, a microtiter plate was coated with an antibody directed against pKe # 83 (eg rabbit anti-pKe # 83 IgG, 1 ⁇ g / well). Then the remaining unspecific binding sites of the microtiter plate were blocked by treatment with 0.1% by weight gelatin in phosphate-buffered saline ("PBS / gelatin").
  • PBS phosphate-buffered saline
  • the microtiter plate was then mixed with different concentrations of the pKe # 83 protein as a calibrator or with dilutions of unknown samples (in which the pKe # 83 concentration should be determined).
  • the plate was incubated with an IgG preparation from a second species (eg with mouse anti-pKe # 83 IgG) (eg with shaking for one hour) Room temperature).
  • the plate was incubated with a peroxidase-labeled commercial rabbit anti-mouse IgG antibody preparation (for example Fc-specific Fab 2- POX from Dianova GmbH, Hamburg).
  • Peroxidase here stands for practically any labeling of the antibody, for example with enzymes, fluorescent molecules or luminescent molecules.
  • the colorless peroxidase substrate ortho-phenylenediamine was added, which is converted into a colored product by the peroxidase activity.
  • the color formation is quantified in a microtiter plate photometer at 490 nm against 405 nm (ordinate).
  • lysates from two different cos-transfectant approaches which differ in the expression of pKe # 83, were tested simultaneously.
  • the cos cells of one approach were transfected with the vector construct pcDNA3.1 / pKe # 83 (approach "Cos pKe # 83") and those of the other approach with the pcDNA3.1 vector without insert (approach "Cos").
  • polyclonal antibodies may as well be monoclonal antibodies that are directed against the protein pKe # 83.
  • monoclonal antibodies that are directed against the protein pKe # 83.
  • indirect approach via a labeled species-specific anti-IgG Antibodies can also be carried out with a directly labeled anti-pKe # 83 antibody respectively.
  • Antisense oligonucleotides are taken up by cells, including keratinocytes (see: G. Hartmann et el. 1998: Antisense oligonucleotides, Guides ⁇ videblatt 95, number 24, C1115 - C1119 ). They bind specifically to the mRNA present in the cell and inhibit its translation and thus the expression of the corresponding protein (see: Y.-S. Lee et el. 1997, Definition by specific antisense oligonucleotides of a role for protein kinase C ⁇ in expression of differentiation markers in normal and neoplastic mouse epidermal keratinocytes, Molecular Carcinogenesis 18: 44-53 ).
  • Suitable antisense oligonucleotides were produced using the pKe # 83-specific nucleotide sequence ( SEQ ID NO: 1 or SEQ ID NO: 7 ). They were adjusted to a concentration of 100 ⁇ M with a suitable buffer medium (so-called "oligo buffer”).
  • oligo buffer a suitable buffer medium
  • HaCaT cells were cultivated at 37 ° C. and 7% CO 2 to a confluency of 70-80%. The cells were trypsinized (10 minutes 0.2% by weight EDTA, 0.1% by weight trypsin, 5-10 minutes) and adjusted to a concentration of 25,000 cells / ml. 100 ⁇ l of cell suspension (corresponding to 2500 cells) were pipetted in per well of a microtiter culture plate (96 wells).
  • the cells were incubated for 1 hour under the aforementioned cultivation conditions, then the antisense oligonucleotide (2 ⁇ l of a 100 ⁇ M solution) was added and the incubation was continued for 24-48 hours.
  • Cell batches served as negative controls, to which oligonucleotides with the same base distribution but a randomly selected sequence were added.
  • FIG. 9 shows HaCaT cells which have been treated with control oligonucleotides
  • FIG. 9.B shows HaCaT cells which have been treated with pKe # 83-specific antisense Oligonucleotides are treated.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Organic Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Biophysics (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Biochemistry (AREA)
  • Immunology (AREA)
  • Toxicology (AREA)
  • Zoology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Peptides Or Proteins (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Medicines Containing Material From Animals Or Micro-Organisms (AREA)
  • Materials For Medical Uses (AREA)
EP99122909A 1998-11-26 1999-11-18 Protéine régulatrice pKe 83 de keratinocytes humains Expired - Lifetime EP1004668B1 (fr)

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
DE19854672 1998-11-26
DE19854672 1998-11-26
DE19856301A DE19856301C1 (de) 1998-11-26 1998-12-07 Regulatorisches Protein pKe#83 aus humanen Keratinozyten
DE19856301 1998-12-07

Publications (3)

Publication Number Publication Date
EP1004668A2 true EP1004668A2 (fr) 2000-05-31
EP1004668A3 EP1004668A3 (fr) 2000-08-30
EP1004668B1 EP1004668B1 (fr) 2005-01-05

Family

ID=26050408

Family Applications (1)

Application Number Title Priority Date Filing Date
EP99122909A Expired - Lifetime EP1004668B1 (fr) 1998-11-26 1999-11-18 Protéine régulatrice pKe 83 de keratinocytes humains

Country Status (6)

Country Link
US (1) US20070154911A1 (fr)
EP (1) EP1004668B1 (fr)
AT (1) ATE286535T1 (fr)
AU (1) AU3030500A (fr)
CA (1) CA2347850A1 (fr)
WO (1) WO2000031125A2 (fr)

Non-Patent Citations (7)

* Cited by examiner, † Cited by third party
Title
EMBL DATABASE; EMHUM1.AB020710; ACCESSION-NO.: AB020710, 9. Februar 1999 (1999-02-09), XP002140479 & NAGASE, T. ET AL.: "Prediction of the coding sequences of unidentified human genes. XII. The complete sequences of 100 new cDNA clones from brain which code for large proteins in vitro" DNA RESEARCH, Bd. 5, Dezember 1998 (1998-12), Seiten 355-364, XP002111974 *
HILLIER, L. ET AL.: "WashU-Merck EST Project 1997" EMBL DATABASE; EMEST28.HS1214076; ACCESSION-NO.: AA418645, 14. Mai 1997 (1997-05-14), XP002140481 *
HILLIER, L. ET AL.: "WashU-NCI human EST Project" EMBL DATABASE; EMEST7.AI205014; ACCESSION-NO.: AI205014, 19. Oktober 1998 (1998-10-19), XP002140480 *
JAEGER, C.: "Identification of a novel transmembrane-protein (pKe#192/MAP17) expressed by human keratinocytes in the upper stratum granulosum of the epidermis" JOURNAL OF INVESTIGATIVE DERMATOLOGY, Bd. 112, Nr. 4, April 1999 (1999-04), Seite 574 XP000909987 *
JIANG, C.-K., ET AL.: "Epidermal growth factor and transforming growth factor alpha specifically induce the activation- and hyperproliferation-associated keratins 6 and 16" PROC.NATL.ACAD.SCI.USA, Bd. 90, Nr. 14, 1993, Seiten 6786-6790, XP000910179 *
KNAPP, B. ET AL.: "Three cDNA sequences of mouse type I keratins" JOURNAL OF BIOLOGICAL CHEMISTRY, Bd. 262, Nr. 2, 1987, Seiten 938-945, XP000910011 *
KOMINE, M. ET AL.: "The activated keratinocytes" ACTA DERMATOVENEROLOGICA A.P.A., Bd. 4, Nr. 4, 1995, Seiten 169-173, XP000915531 *

Also Published As

Publication number Publication date
WO2000031125A2 (fr) 2000-06-02
AU3030500A (en) 2000-06-13
US20070154911A1 (en) 2007-07-05
EP1004668A3 (fr) 2000-08-30
WO2000031125A3 (fr) 2000-10-19
EP1004668B1 (fr) 2005-01-05
ATE286535T1 (de) 2005-01-15
CA2347850A1 (fr) 2000-06-02

Similar Documents

Publication Publication Date Title
DE19964269B4 (de) Verfahren zur Detektion des Proliferationspotentials von Zellen
DE69233421T2 (de) Neuartige rezeptor-typ tyrosinkinase und deren verwendung
DE69325383T2 (de) Testreihenanalyse-Verfahren mit transkriptionsfaktor DP-1
DE69808743T2 (de) Netrinrezeptoren
DE69229219T2 (de) Neuartige, physiologisch aktive substanz epimorphin, für sie kodierende gene und antikörper gegen epimorphin
EP0875567A2 (fr) Protéines à doigts de zinc se liant à l'oncogène mycm ainsi comment leur préparation et utilisation
DE19957065B4 (de) Screening-Verfahren für Arzneistoffe
EP0805204B1 (fr) Protéine récepteur spécifique de l'épididyme et son utilisation
DE69132682T2 (de) Zellwachstumsinhibitoren
EP1114157A2 (fr) Proteine regulatrice issue de keratinocytes humains
DE69513025T2 (de) Test für inhibitoren von dp-1 und anderen dp-proteinen
DE19856301C1 (de) Regulatorisches Protein pKe#83 aus humanen Keratinozyten
EP1004668B1 (fr) Protéine régulatrice pKe 83 de keratinocytes humains
DE69530261T3 (de) Durch Zelldichte stimulierte Protein-Tyrosin-Phosphatasen
EP1072681B1 (fr) Protéine regulatrice pKe#165 des kératinocytes humaines
EP1007671B1 (fr) Gene ii de fanconi
DE69832447T2 (de) Gen kodierend für Afadin-1
DE10139874A1 (de) Humane Mater-Proteine
EP1023445B1 (fr) Facteur de croissance derive de la cadherine et son utilisation
DE19964386B4 (de) Verfahren zur Herstellung von Makrophagenmigrations-Inhibitionsfaktor (MIF)
EP1556488B1 (fr) Hf-chondroosteomoduline, production et utilisation pour realiser le traitement ou le diagnostic de maladies des os ou des cartilages, de l'adipose, de maladies inflammatoires et de maladies de la peau
WO1999042477A2 (fr) Transporteur pour medicaments a couplage saccharidique
DE10001377A1 (de) bHLH-Transkriptionsfaktor ASCL3 und diesen enthaltendes Arzneimittel
DE4236358A1 (de) Nachweis und Inhibierung von Malatenzym in Tumorzellen
DE60038531T2 (de) Transporter organischer anionen der plazenta und dessen gen

Legal Events

Date Code Title Description
PUAI Public reference made under article 153(3) epc to a published international application that has entered the european phase

Free format text: ORIGINAL CODE: 0009012

AK Designated contracting states

Kind code of ref document: A2

Designated state(s): AT BE CH CY DE DK ES FI FR GB GR IE IT LI LU MC NL PT SE

AX Request for extension of the european patent

Free format text: AL;LT;LV;MK;RO;SI

PUAL Search report despatched

Free format text: ORIGINAL CODE: 0009013

AK Designated contracting states

Kind code of ref document: A3

Designated state(s): AT BE CH CY DE DK ES FI FR GB GR IE IT LI LU MC NL PT SE

AX Request for extension of the european patent

Free format text: AL;LT;LV;MK;RO;SI

17P Request for examination filed

Effective date: 20000810

17Q First examination report despatched

Effective date: 20001027

AKX Designation fees paid

Free format text: AT BE CH CY DE DK ES FI FR GB GR IE IT LI LU MC NL PT SE

AXX Extension fees paid

Free format text: AL PAYMENT 20000810;LT PAYMENT 20000810;LV PAYMENT 20000810;MK PAYMENT 20000810;RO PAYMENT 20000810;SI PAYMENT 20000810

GRAP Despatch of communication of intention to grant a patent

Free format text: ORIGINAL CODE: EPIDOSNIGR1

GRAS Grant fee paid

Free format text: ORIGINAL CODE: EPIDOSNIGR3

GRAA (expected) grant

Free format text: ORIGINAL CODE: 0009210

AK Designated contracting states

Kind code of ref document: B1

Designated state(s): AT BE CH CY DE DK ES FI FR GB GR IE IT LI LU MC NL PT SE

AX Request for extension of the european patent

Extension state: AL LT LV MK RO SI

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: NL

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20050105

Ref country code: IT

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT;WARNING: LAPSES OF ITALIAN PATENTS WITH EFFECTIVE DATE BEFORE 2007 MAY HAVE OCCURRED AT ANY TIME BEFORE 2007. THE CORRECT EFFECTIVE DATE MAY BE DIFFERENT FROM THE ONE RECORDED.

Effective date: 20050105

Ref country code: IE

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20050105

Ref country code: FI

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20050105

REG Reference to a national code

Ref country code: GB

Ref legal event code: FG4D

Free format text: NOT ENGLISH

REG Reference to a national code

Ref country code: CH

Ref legal event code: EP

REG Reference to a national code

Ref country code: IE

Ref legal event code: FG4D

Free format text: GERMAN

REF Corresponds to:

Ref document number: 59911400

Country of ref document: DE

Date of ref document: 20050210

Kind code of ref document: P

REG Reference to a national code

Ref country code: CH

Ref legal event code: NV

Representative=s name: PATENTANWALT DIPL. ING. WOLFGANG HEISEL

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: SE

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20050405

Ref country code: GR

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20050405

Ref country code: DK

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20050405

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: ES

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20050416

GBT Gb: translation of ep patent filed (gb section 77(6)(a)/1977)

Effective date: 20050519

LTIE Lt: invalidation of european patent or patent extension

Effective date: 20050105

NLV1 Nl: lapsed or annulled due to failure to fulfill the requirements of art. 29p and 29m of the patents act
REG Reference to a national code

Ref country code: IE

Ref legal event code: FD4D

PLBE No opposition filed within time limit

Free format text: ORIGINAL CODE: 0009261

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: NO OPPOSITION FILED WITHIN TIME LIMIT

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: CY

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20051118

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: MC

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20051130

Ref country code: LU

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20051130

26N No opposition filed

Effective date: 20051006

ET Fr: translation filed
REG Reference to a national code

Ref country code: GB

Ref legal event code: FG4D

Free format text: NOT ENGLISH

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: PT

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20050605

PGFP Annual fee paid to national office [announced via postgrant information from national office to epo]

Ref country code: DE

Payment date: 20071130

Year of fee payment: 9

Ref country code: BE

Payment date: 20070924

Year of fee payment: 9

PGFP Annual fee paid to national office [announced via postgrant information from national office to epo]

Ref country code: CH

Payment date: 20071123

Year of fee payment: 9

Ref country code: AT

Payment date: 20071130

Year of fee payment: 9

PGFP Annual fee paid to national office [announced via postgrant information from national office to epo]

Ref country code: GB

Payment date: 20071127

Year of fee payment: 9

Ref country code: FR

Payment date: 20070926

Year of fee payment: 9

REG Reference to a national code

Ref country code: CH

Ref legal event code: PCAR

Free format text: PATENTANWALT DIPL.-ING. (UNI.) WOLFGANG HEISEL;HAUPTSTRASSE 14;8280 KREUZLINGEN (CH)

BERE Be: lapsed

Owner name: *KRAMER MICHAEL

Effective date: 20081130

REG Reference to a national code

Ref country code: CH

Ref legal event code: PL

GBPC Gb: european patent ceased through non-payment of renewal fee

Effective date: 20081118

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: AT

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20081118

REG Reference to a national code

Ref country code: FR

Ref legal event code: ST

Effective date: 20090731

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: BE

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20081130

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: LI

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20081130

Ref country code: DE

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20090603

Ref country code: CH

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20081130

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: GB

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20081118

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: FR

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20081130